Compositions and Methods of Treating Muscular Dystrophy with Thromboxane-A2 Receptor Antagonists

ABSTRACT

The present invention is directed to methods of treating and/or ameliorating muscular dystrophy and/or treating cardiomyopathy in muscular dystrophy patients by administration of a therapeutically effective amount of a thromboxane A 2  receptor antagonist.

This invention was made with government support under grant numbersR01HL095797 and P01HL108800 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the use of thromboxane A₂ receptorantagonists (e.g., Ifetroban) in the treatment of muscular dystrophy inmammals, e.g., humans, and pharmaceutical compositions for the samecomprising thromboxane A₂ receptor antagonists (e.g., Ifetroban) in aneffective amount to treat these diseases.

BACKGROUND OF THE INVENTION

Muscular Dystrophy (MD) is a group of 30+ diseases that causesprogressive weakness and loss of muscle mass due to mutations indystrophin, a protein needed to form healthy muscle, Duchenne MD (DMD)comprises half of MD; affects 1 in 3,500 boys and ⅓ have no familyhistory. Onset is between ages 2 and 3 and progresses rapidly. Becker MD(BMD) is the 2nd most common form of MD; 1 in 30,000 boys; BMD is milderand slowly progresses compared to DMD; symptoms may not he seen untilteens, mid-20s or later. Limb-Girdle MD (LGMD) can affects as many as 1in 14,500 and causes weakness and wasting of the muscles in the proximalarms and legs.

Complications of muscular dystrophy include inability to walk, breathingproblems, scoliosis, cardiomyopathy and swallowing problems. There is nocure. Treatment to-date is to manage symptoms or slow progression.

Delta-sarcoglycan (DSG) is a transmembrane glycoprotein which forms as acomplex, the dystrophin-associated glycoprotein complex (DGC). The IGCplays a central role in maintaining integrity of the cell membrane bylinking the extracellular matrix (“ECM”; a substance containingcollagen, elastin, proteoglycans, glycosaminoglycans, and fluid,produced by cells and in which the cells are embedded) and cytoskeleton(the inner structural elements, or backbone, of a cell. It consists ofmicrotubules and various filaments that spread out through thecytoplasm, providing both structural support and a means of transportwithin the cell).

In both skeletal and cardiac muscle, the DGC consists of dystrophin, thesyntrophins, a- and b-dystroglycan (a-, b-DG), the sarcoglycans (a-, b-,g-, d-SG), and sarcospan (SSPN).

Mutations in the dystrophin gene lead to high incidence ofcardiomyopathy in DMD and BMD. Mutations in sarcoglycans within DGC areresponsible for Limb-Girdle MD and associated with cardiomyopathy. Amajor function of dystrophin is to strengthen the sarcolemma bycross-linking the ECM with the cytoskeleton. Utrophin and a7b1 integrinfulfil the same function. Dystrophin works to connect sarcolemma tocytoplasmic actin cytoskeleton. Dysfunction produces membraneinstability, elevated [Ca2+]I and disrupted NO signaling. γ- and δ-SGform a core necessary for delivery/retention of other SG to themembrane.

Patients with mutations in DSG (e.g., patients suffering from musculardystrophy) present with cardiomyopathy.

Absence of dystrophin in Duchenne muscular dystrophy (DMD) causesprogressive breakdown of muscle cells. In the heart, loss of dystrophinleads to abnormally increased intracellular calcium, degradation ofcontractile proteins, fibrosis, and myocardial death. With advances inrespiratory support, cardiomyopathy is now a primary cause of deathamongst DMD patients. DMD patients develop an insidious decline incardiac function leading to heart failure and can also developarrhythmias, with the potential for sudden cardiac death, even withminimal decrease in cardiac function by physical symptoms orechocardiography. Because of this, cardiac magnetic resonance (CMR) isuseful for detection of early cardiac involvement in DMD patients.Increased myocardial fibrosis and expanded extracellular volume in CMRpredicts left ventricular (LV) dysfunction, and are associated with anincreased risk of arrhythmia and hospitalization for heart failure ordeath.

While less severely affected than skeletal and cardiac muscle,intestinal smooth muscle function can also be altered by atrophy andfibrosis. In DMD patients, particularly when wheelchair-bound, this canlead to poor gut motility, gastroesophageal reflux, and chronicconstipation, which negatively affect patient quality of life. Morecritically, the possible complications of dilatation, fecal impaction,or intestinal pseudo-obstruction can be life-threatening.

The cellular damage characteristic of DMD is also associated withincreased formation of reactive oxygen species, or oxidative stress.(Grosso, et al., Isoprostanes dystrophinopathy: Evidence of increasedoxidative stress. Brain Dev. 2008, 30(6):391-5,doi:10.1016/j.braindev.2007.11.005, PubMed PMID: 18180123). These freeradicals can react with membrane phospholipids to form isoprostanes,which circulate lively after release by phospholipase, and therelatively stable 15-F2t-isoprostane (F2-IsoP) is a primary biomarker ofin vivo oxidative stress. (Montuschi, et al., Isoprostanes: markers andmediators of oxidative stress. FASEB J, 2004; 18(15):1791-800. doi:10.1096/fj.04-2330rev). Plasma F2-IsoP levels are increased in DMDpatients (Grosso, et al., cited above), and urinary F2-IsoP levels areincreased in heart failure patients, where they correlate with theseverity of the disease (Cracowski, et al., Increased formation ofF(2)-isoprostanes in patients with severe heart failure. Heart. 2000;84(4):439-40. PubMed PMID:10995421; PMCID: PMC172944614). In addition toheralding cellular stress, isoprostanes can also be the source of damagevia activation of the thromboxane/prostanoid receptor (TPr), and F2-IsoPsignaling through the TPr decreases angiogenesis and causesvasoconstriction (Bauer, et al., Pathophysiology of isoprostanes in thecardiovascular system: implications of isoprostane-mediated thromboxaneA2 receptor activation. Brit J Pharmacol. 2014; 171:3115-3115) andfibrosis (Acquaviva, et al. Signaling pathways involved inisoprostane-mediated fibrogenic effects in rat hepatic stellate cells.Free Radic Biol Med. 2013; 65:201-7,doi:10.1016/j.freeradbiomed.2013.06.023. PubMed PMID: 23792773;Comporti, et al. Isoprostanes and hepatic fibrosis, Mol Aspects Med.2008; 29(1-2):43-9. doi: 10.1016/j.mam.2007.09.011. PubMed PMID:18061254).

Fibrosis is the formation of excess fibrous connective tissue in anorgan or tissue in a reparative or reactive process. This can be areactive, benign, or pathological state, and physiologically acts todeposit connective tissue, which can obliterate the architecture andfunction of the underlying organ or tissue. Fibrosis can be used todescribe the pathological state of excess deposition of fibrous tissue,as well as the process of connective tissue deposition in healing. Whilethe formation of fibrous tissue is normal, and fibrous tissue is anormal constituent of organs or tissues in the body, scarring caused bya fibrotic condition may obliterate the architecture of the underlyingorgan or tissue.

To date, there are no commercially available therapies that areeffective in treating or preventing fibrotic disease. Conventionaltreatment frequently involves corticosteroids, such as prednisone,and/or other medications that help improve muscle strength and delay theprogression of certain types of muscular dystrophy. Also, heartmedications, such as angiotensin-converting enzyme (ACE) inhibitors orbeta blockers may be administered to muscular dystrophy patients, if themuscular dystrophy damages the heart.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new methods oftreating muscular dystrophy in mammals, e.g., humans.

In accordance with the above objects, the present invention provides formethods of treating muscular dystrophy by administering, atherapeutically effective amount of a thromboxane A₂ receptor antagonistto a patient in need thereof.

In accordance with the above objects and others, the present inventionis directed in part to a method of treating or ameliorating musculardystrophy in a subject in need of treatment thereof, comprisingadministering a therapeutically effective amount of a thromboxane A2receptor antagonist to the patient. The muscular dystrophy is fibrosisis selected from the group consisting of Duchenne MD (DMD). Becker MD,and Limb-Girdle MD. The thromboxane A2 receptor antagonist may beadministered orally, intranasally, rectally, vaginally, sublingually,buccally, parenterally, or transdermally. In certain preferredembodiments, the method further comprises administering the thromboxaneA2 antagonist to the patient: on a chronic basis. In certainembodiments, the thromboxane A₂ receptor antagonist comprises atherapeutically effective amount of[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2]hept-2-yl]methyl]-benzenepropanoicacid (Ifetroban), and pharmaceutically acceptable salts thereof. Incertain embodiments, the thromboxane A₂ receptor antagonist comprises atherapeutically effective amount of[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt (Ifetroban Sodium). In certain preferredembodiments, the cardiac function of the patient is maintained orimproved. Certain embodiments of the invention are directed to themethod, wherein the thromboxane A2 receptor antagonist is administeredprophylactically to prevent cardiomyopathy in the patient, and/or toprophylactically to prevent gastrointestinal dysfunction in the patient.In certain preferred embodiments, the therapeutically effective amountis from about 50 mg to about 500 mg. In certain preferred embodiments,the thromboxane A2 receptor antagonist is ifetroban and thetherapeutically effective amount is from about 150 mg to about 350 mgper day. In certain embodiments, the ifetroban is administered orally.In certain embodiments, the present invention is directed to a method oftreating and/or ameliorating muscular dystrophy in a patient in needthereof, comprising administering to a patient in need thereof atherapeutically effective amount of a thromboxane A₂ receptor antagonistto provide a desired plasma concentration of the thromboxane A₂ receptorantagonist of about 0.1 ng/ml to about 10,000 ng/ml.

The invention is also directed to a method of providing cardioprotectiveeffects to a human patient(s) suffering from muscular dystrophy via theadministration of a thromboxane A₂ receptor antagonist as describedherein.

The invention is further directed to a method a improving right heartadaptation to load stress in a human patient(s) suffering from musculardystrophy via the administration of a thromboxane A₂ receptor antagonistas described herein.

The invention is further directed to a method of treating cardiac and/orgastrointestinal dysfunction in a human patient suffering from musculardystrophy, comprising chronically administering a therapeuticallyeffective amount of a thromboxane A2 receptor antagonist to the humanpatient. In certain preferred embodiments, the thromboxane A2 receptorantagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid (Ifetroban), and pharmaceutically acceptable salts thereof, and incertain most preferred embodiments the thromboxane A2 receptorantagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt (Ifetroban Sodium). The therapeutically effectiveamount may be, e.g., from about 100 mg to about 500 mg. The thromboxaneA2 receptor antagonist may be administered, e.g., in an amount fromabout 50 or 100 mg to about 500 mg per day. In certain embodiments, thethromboxane A2 receptor antagonist is ifetroban or a pharmaceuticallyacceptable salt thereof and the daily dose is from about 150 mg to about350 mg per day. In certain embodiments, the ifetroban is administeredorally. In certain embodiments, the gastrointestinal dysfunction issmooth muscle dysfunction. In certain embodiments, the therapeuticallyeffective amount of ifetroban provides improved ventricular function tothe heart of the patient.

The present invention also relates to methods and compositions fortreating muscular dystrophy in a mammal(s) or human(s) in need oftreatment thereof, the method comprising administering a therapeuticallyeffective amount of a thromboxane A₂ receptor antagonist to a subject(s)or patient(s) in need thereof. Preferably, the method of treatmentcomprises administering a composition comprising administering atherapeutically effective amount of a thromboxane A₂ receptor antagonistto a muscular dystrophy patient in need thereof in an amount effectiveto improve heart function. Further provided is a method of preventingfibrosis or sclerosis in a subject(s) or patient(s) in need of suchtreatment, comprising administering a composition comprising athromboxane A₂ receptor antagonist in an amount effective to reduce theformation of fibrotic or sclerotic tissue that would occur in theabsence of such treatment.

In a certain embodiment, the fibrosis is associated with afibroproliferative disease selected from the group consisting of heartfibrosis, kidney fibrosis, liver fibrosis, lung fibrosis, and systemicsclerosis.

BRIEF DESCRIPTION A THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1A is a photograph of a vehicle-treated DKO (double knockout) Mouseat 10 weeks;

FIG. 1B is a photograph of an ifetroban-treated DKO mouse at 10 weeks;

FIG. 2 is a graph showing plasma cTNI in dSG KO males at 3 months(vehicle-treated versus ifetroban-treated);

FIG. 3 is a graph showing 3 month Echo data in mice (WT(wild-type),dSG-vehicle and dSG-ifetroban treated);

FIG. 4 is a graph providing cardiac output data for male mice at 3months (WT, dSG KO-vehicle and dSG KO-ifetroban treated);

FIG. 5 is a graph providing spontaneous exercise data for 6 month ofmales (WT, dSG-vehicle and dSG-ifetroban treated);

FIG. 6 is a graph showing average wire hang time in male mice at 6months (WT, dSG-vehicle and dSG-ifetroban treated);

FIG. 7 is a graph showing the results of a wire hanging experiment(average hang time) at 6 months (WT, dSG; vehicle versusifetroban-treated; P=0.0056 for genotype by 2-way ANOVA);

FIG. 8 is a graph showing 6 month wire bang time (longest time) for malemice tested (WT, dSG-vehicle, dSG-ifetroban treated);

FIGS. 9A (dSGKO-vehicle) and 9B (DsGKO-ifetroban) show cardiac histologyin dSG KO males. Less fibrosis seen in ifetroban treated RV. Shown isMasson's trichrome at 4× for gross histology. All tears/folds/redhotspots from slice preparation and not pathology. Some RV may also beaffected by slicing (arrows).

FIGS. 10A(dSG-Veh), 10B(dSG-veh), 10C(dSG-ifetroban) and10D(dSG-ifetroban) show cardiac histology in dSG KO males (usingMasson's trichrome, 2×). It can be seen that there is less fibrosis inthe ifetroban treated RV. RV=right ventricle.

FIGS. 11A1, 11A2, 11A3 and 11A4 shows cardiac histology in dSG KO males(using Masson's trichrome, 10×) in the left ventricle (11A1=mouse #1,dSG KO-vehicle; 11A2=mouse #2, dSG KO-vehicle; 11A3=mouse #1, dSGKO-ifetroban; and 11A4=mouse #2, dSG KO-ifetroban); FIGS. 11B1, 11B2,11B3 and 11B4 shows cardiac histology in the right ventricle (11B1=mouse#1, dSG KO-vehicle; 11B2=mouse #2, dSG KO-vehicle; 11B3=mouse #1. dSGKO-ifetroban; and 11B4-mouse #2, dSG KO-ifetroban). LV=left ventricle;RV=right ventricle. Less fibrosis was seen in ifetroban-treated KO mice.

FIGS. 12A(WT1), 12B(dSG-KO-vehicle), 12C(WT2) and 12D(dSG-KO-ifetroban)shows skeletal muscle histology in WT and dSG KO males (tibialiscross-section, using Masson's trichrome). Some fibrosis ma be due tospecific section of muscle.

FIGS. 13A(WT-vehicle), 13B(WT-ifetroban), 13C(dSG KO-vehicle) and13D(dSG-KO-ifetroban) are cross-sections of intestinal tissue showingthat ifetroban may prevent the loss of intestinal smooth muscle in thelarge intestine Muscularis. The DSG KO mice were missing smooth muscle(especially missing longitudinal smooth muscle) while ifetroban-treatedmice have similar sections to WT smooth muscle. “H&E”=Hematoxylin &eosin. FIG. 13 shows that ifetroban-treated dSG KO mice have lessfibrosis than vehicle-treated dSG KO mice.

FIGS. 14A and 14B are graphs showing the percent survival of dSG KOmales (14A) and dSG females (14B) treated with ifetroban or vehicle.

FIG. 15 are graphs showing wire hang in WT and DKO males at 10 weeks(ifetroban-treated (“ife”) versus vehicle);

FIG. 16 is a graph showing spontaneous running in WT and DKO micemeasured from 9-10 weeks (DKO-vehicle and DKO-ifetroban treated); and

FIG. 17 is a graph showing survival for all DKO mice (vehicle andifetroban treated).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the above stated objects, it is believed thatadministration of a therapeutically effective amount of a thromboxane A₂receptor antagonist to a subject(s) or patient(s) in need thereof cantreat cardiomyopathy associated with muscular dystrophy.

The phrase “therapeutically effective amount” refers to that amount of asubstance that produces some desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. The effectiveamount of such substance will vary depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart.

The TPr is a G protein-coupled receptor which is located in platelets,immune cells, smooth muscle, and cardiomyocytes, and its activation hasdeleterious consequences in the heart. We have recently shown (in ourU.S. Patent Application Publication No. 2015/0328190) that blockade ofthe TPr with the antagonist ifetroban dramatically decreases rightventricular fibrosis and improves cardiac function in apressure-overload model of pulmonary arterial hypertension. Although theTPr has multiple endogenous ligands including F2-IsoP, thromboxane A2,prostaglandin H2, and 20-HETE, blockade of thromboxane synthase withozagrel or prostaglandin/thromboxane synthesis with aspirin had noeffect on fibrosis or cardiac function in our pressure-overload model.Thus, F2-IsoP is an excellent candidate as an activating ligand of theTPr in the stressed heart. Beyond the right ventricle TPr activationalso contributes to LV hypertrophy and heart failure in mouse models ofsystemic hypertension and Gh-overexpression. In addition, TPr activationcauses increased intracellular calcium, arrhythmia, and cell death inventricular cardiomyocytes, and decreased peristalsis in the gut.Although the role of the TPr in MD is unknown, these actions positionthe receptor to have an impact on some of the most pressing concerns inDMD.

Applicants explored the possibility that TPr activity may contribute topathology in muscular dystrophy. In preliminary studies, the effects ofblocking TPr activity in a δ-sarcoglycan knockout (dSG KO) mouse modelof limb-girdle muscular dystrophy (LGMD). We found that treatment withthe antagonist ifetroban, given in drinking water, limits the formationof cardiac fibrosis and prevents a decline in cardiac function whilenormalizing elevated plasma cardiac troponin I levels, a clinically-usedbiomarker for cardiac injury. The inhibition of LV epicardial fibrosismay have particular applicability to DMD patients, where cardiacfibrosis typically begins in the sub-epicardium of the left ventricular(LV) free wall and progresses to include the remaining LV free wall andseptum. Ifetroban treatment also significantly improved survival in dSGKO mice, and in utrophin/dystrophin double knockout (DKO) mice, a modelof severe DMD, TPr antagonism with ifetroban improved 10-week survivalfrom 56% to 100%. Therefore, it is believed that TPr activitycontributes to pathology in muscular dystrophy.

In accordance with the present invention, it is believed that increasedisoprostane signaling through the TPr contributes to cardiomyopathy andsmooth muscle dysfunction in DMD, and thus treatment with ifetroban, anorally active TPr antagonist, will improve cardiac and gut function anddecrease spontaneous mortality in mammals (as demonstrated inpreclinical mouse models of DMD). It is also believed that treatmentwith a thromboxane receptor antagonist (ifetroban) may contribute tocardioprotection by increasing the regenerative capability of the heart,and therefore may provide functional improvement of the heart (e.g.,improved ventricular function). Thus, the invention is directed in partto the use of TPr antagonists as a treatment for cardiac and/orgastrointestinal dysfunction in DMD. The invention is also directed inpart to the use of TPR antagonists for providing cardioprotection byincreasing the regenerative capability of the heart and/or providingfunctional improvement of the heart of a muscular dystrophy (human)patient.

The term “thromboxane A2 receptor antagonist” as used herein refers to acompound that inhibits the expression or activity of a thromboxanereceptor by at least or at least about 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in astandard bioassay or in vivo or when used in a therapeutically effectivedose. In certain embodiments, a thromboxane A2 receptor antagonistinhibits binding of thromboxane A₂ to the receptor. Thromboxane A2receptor antagonists include competitive antagonists (i.e., antagoniststhat compete with an agonist for the receptor) and non-competitiveantagonists. Thromboxane A2 receptor antagonists include antibodies tothe receptor. The antibodies may be monoclonal. They may be human orhumanized antibodies. Thromboxane A2 receptor antagonists also includethromboxane synthase inhibitors, as well as compounds that have boththromboxane A2 receptor antagonist activity and thromboxane synthaseinhibitor activity.

Thromboxane A₂ Receptor Antagonist

The discovery and development of thromboxane A₂ receptor antagonists hasbeen an objective of many pharmaceutical companies for approximately 30years (see, Dogne J-M, et al., Exp. Opin. Ther. Patents 11: 1663-1675(2001)). Certain individual compounds identified by these companies,either with or without concomitant thromboxane synthase inhibitoryactivity, include ifetroban (BMS), ridogrel (Janssen), terbogrel (BI),UK-147535 (Pfizer), GR 32191 (Glaxo), and S-18886 (Servier). Preclinicalpharmacology has established that this class of compounds has effectiveantithrombotic activity obtained by inhibition of the thromboxanepathway. These compounds also prevent vasoconstriction induced bythromboxane A₂ and other prostanoids that act on the thromboxane A₂receptor within the vascular bed, and thus may be beneficial for use inpreventing and/or treating hepatorenal syndrome and/or hepaticencephalopathy.

Suitable thromboxane A2 receptor antagonists for use in the presentinvention may include, for example, but are not limited to smallmolecules such as ifetroban (BMS;[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(pentylamino)carbony-1]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2yl]methyl[benzenepropanoicacid), as well as others described in U.S. Patent ApplicationPublication No. 2009/0012115, the disclosure of which is herebyincorporated by reference in its entirety.

Additional thromboxane A2 receptor antagonists suitable for use hereinare also described in U.S. Pat. Nos. 4,839,384 (Ogletree); U.S. Pat. No.5,066,480 (Ogletree, et al.); U.S. Pat. No. 5,100,889 (Misra, et al.);U.S. Pat. No. 5,312,818 (Rubin, et al.); U.S. Pat. No. 5,399,725 (Poss,et al.), and U.S. Pat. No. 6,509,348 (Ogletree), the disclosures ofwhich are hereby incorporated by reference in their entireties. Thesemay include, but are not limited to, interphenylene 7-oxabicyclo-heptylsubstituted heterocyclic amide prostaglandin analogs as disclosed inU.S. Pat. No. 5,100,889, including:

[1S-(1α,2α,3α,4α)]-2-[[3-[4-[[(4-cyclo-hexylbutyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]-hept-2-yl]methyl]benzenepropanoicacid (SQ 33,961) or esters or salts thereof;

[1S-(1α,2α,3α,4α)]-2-[[3-[4-[[[(4-chloro-phenyl)-butyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]benzenepropanoicacid or esters, or salts thereof;

[1S-(1α,2α,3α,4α)]-3-[[3-[4-[[(4-cycloh-exylbutyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]benzeneacetic acid, or esters or salts thereof;

[1S-(1α,2α,3α,4α)]-[2-[[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]phenoxy]aceticacid, or esters or salts thereof;

[1S-(1α,2α,3α,4α]-[2-[[3-[4-[[(7,7-dime-thyloctyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-methyl]benzenepropanoicacid, or esters or salts thereof.

7-oxabicycloheptyl substituted heterocyclic amide prostaglandin analogsas disclosed in U.S. Pat. No. 5,100,889, issued Mar. 31, 1992, including[1S-[1α,2α(Z),3α,4α)]-6-[3-[4-[[(4-cyclohexylbutyl)amino]-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-thiazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)methylamino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[(1-pyrrolidinyl)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[(cyclohexylamino)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl-4-hexenoicacid or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(2-cyclohexyl-ethyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[[2-(4-chloro-phenyl)ethyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]-6-[3-[4-[[(4-chlorophenyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[[[4-(4-chloro-phenyl)butyl]amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[11α,2α(Z),3α,4α)]]-6-[3-[4.alpha.-[[-(6-cyclohexyl-hexyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters, or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(6-cyclohexyl-hexyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[(propylamino)-carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-butylphenyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[(2,3-dihydro-1H-indol-1-yl)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-N-(phenylsulfonyl)-4-hexenamide;

[1S-[11α,2α(Z),3α,4α)]]-6-[3-[4-[[((4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-N-(methylsulfonyl)-7-oxabicyclo[2-.2.1]hept-2-yl]-4-hexenamide;

[1S-[1α,2α(Z),3α,4α)]]-7-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid, or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-1H-imidazol-2-yl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid or esters or salts thereof;

[1S-[1α,2α,3α,4α)]]-6-[3-[4-[[(7,7-dimethyloctyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

[1S-[1α,2α(E),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid;

[1S-[1α,2α,3α,4αa)]-3-[4-[[(4-cyclohexylbutyl)-amino]carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]heptane-2-hexanoicacid or esters or salts thereof;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-2-oxazolyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-4-hexenoicacid, or esters or salts thereof;

7-oxabicycloheptane and 7-oxabicycloheptene compounds disclosed in U.S.Pat. No. 4,537,981 to Snitman et al, the disclosure of which is herebyincorporated by reference in its entirety, such as[1S-(1α,2α(Z),3α(1E,3S*,4R*),4α)]]-7-[3-(3-hydroxy-4-phenyl-1-pentenyl)-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid (SQ 29,548); the 7-oxabicycloheptane substituted aminoprostaglandinanalogs disclosed in U.S. Pat. No. 4,416,896 to Nakane et al, thedisclosure of which is hereby incorporated by reference in its entirety,such as[1S-[1α,2α(Z),3α,4α)]]-7-[3-[[2-(phenylamino)carbonyl]-hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid: the 7-oxabicycloheptane substituted diamide prostaglandin analogsdisclosed in U.S. Pat. No. 4,663,336 to Nakane et al, the disclosure ofwhich is hereby incorporated by reference in its entirety, such as,[1S-[1α,2α(Z),3α,4α)]]-7-[3-[[[[(1-oxoheptyl)amino]-acetyl]amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoicacid and the corresponding tetrazole, and[1S-[1α,2α(Z),3α,4α)]]-7-[3-[[[[(4-cyclohexyl-1-oxobutyl)-amino]acetyl]amino]methyl]-7-oxabicyclo]2.2.1]hept-2-yl]-5-heptenoicacid;

7-oxabicycloheptane imidazole prostaglandin analogs as disclosed in U.S.Pat. No. 4,977,174, the disclosure of which is hereby incorporated byreference in its entirety, such as[1S-[1α,2α(Z),3α,4α)]]-6-[3-[[4-(4-cyclohexyl-1-hydroxybutyl)-1H-imidazole-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[[4-(3-cyclohexyl-propyl)-1H-imidazol-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α,2α(X(Z),3α,4α)]]-6-[3-[[4-(4-cyclohexyl-1-oxobutyl)-1H-imidazol-1-yl]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester;

[1S-[1α,2α(Z),3α,4α]]-6-[3-(1H-imidazol-1-ylmethyl)-7-oxabicyclo[2.2.1]hept-2-yl]-4-hexenoicacid or its methyl ester; or

[1S-[1α,2α(Z),3α,4α)]]-6-[3-[[4-[[(4-cyclohexyl-butyl)amino]carbonyl]-1H-imidazol-1-yl]methyl-7-oxabicyclo-[2.2.1]-hept-2-yl]-4-hexenoicacid, or its methyl ester;

The phenoxyalkyl carboxylic acids disclosed in U.S. Pat. No. 4,258,058to Witte et al, the disclosure of which is hereby incorporated byreference in its entirety, including4-[2-(benzenesulfamido)ethyl]phenoxy-acetic acid (BM 13,177-BoehringerMannheim), the sulphonamidophenyl carboxylic acids disclosed in U.S.Pat. No. 4,443,477 to Witte et al, the disclosure of which is herebyincorporated by reference in its entirety, including4-[2-(4-chlorobenzenesulfonamido)ethyl]-phenylacetic acid (BM 13,505,Boehringer Mannheim), the arylthioalkylphenyl carboxylic acids disclosedin U.S. Pat. No. 4,752,616, the disclosure of which is herebyincorporated by reference in its entirety, including4-(3-((4-chlorophenyl)sulfonyl)propyl)benzene acetic acid.

Other examples of thromboxane A₂ receptor antagonists suitable for useherein include, but are not limited to vapiprost (which is a preferredexample).(E)-5-[[[(pyridinyl)]3-(trifluoromethyl)phenyl]methylene]amino]-oxy]pentanoicacid also referred to as R68,070-Janssen Research Laboratories,3-[1-(4-chlorophenylmethyl)-5-fluoro-3-methylindol-2-yl]-2.-2-dimethylpropanoicacid [(L-655240 Merck-Frosst) Eur. J. Pharmacol. 135(2): 193, Mar. 17,87],5(Z)-7-([2,4,5-cis]-4-(2-hydroxyphenyl)-2-trifl-uoromethyl-1,3-dioxan-5-yl)heptenoicacid (ICI 185282, Brit. J. Pharmacol. 90 (Proc. Suppl):228 P-Abs, March1987), 5(Z)-7-[2,2-dimethyl-4-phenyl-1,3-dioxan-cis-5-yl]heptenoic acid(ICI 159995, Brit. J. Pharmacol. 86 (Proc. Suppl):808 P-Abs., December1985),N,N′-bis[7-(3-chlorobenzeneamino-sulfony-1)-1,2,3,4-tetrahydro-isoquinolyl]disulfonylimide(SKF 88046, Pharmacologist 25(3): 116 Abs., 117 Abs. August 1983),(1.alpha.(Z)-2.beta.,5.alpha.]-(+)-7-[5-[[(1,1′-biphenyl)-4-yl]-methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl)-4-heptenoicacid (AH 23848-Glaxo, Circulation 72(6): 1208, December 1985,levallorphan allyl bromide (CM 32,191 Sanofi, Life Sci. 31 (20-21):2261,Nov. 15, 1982),(Z,2-endo-3-oxo)-7-(3-acetyl-2-bicyclo[2.2.1]heptyl-5-hepta-3Z-enoicacid, 4-phenyl-thiosemicarbazone (EP092-Univ. Edinburgh, Brit. J.Pharmacol. 84(3):595, March 1985); GR 32,191(Vapiprost)-(1R-[1.alpha.(Z), 2.beta, 3.beta.,5.alpha.]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoicacid; ICI192,605-4(Z)-6-[(2,4,5-cis)2-(2-chlorophenyl)-4-(2-hydroxyphenyl)-1,3-dioxan-5-yl]hexenoicacid; BAY u 3405(ramatroban)-3-[[(4-fluorophenyl)-sulfonyl]amino]-1,2,3,4-tetrahydro-9H-c-arbazole-9-propanoicacid; or ONO 3708-7-[2.alpha.,4.alpha.-(dimethylmethano)-6.beta.-(2-cyclopentyl-2.beta.-hydroxyacetami-do)-1.alpha.-cyclohexyl]-5(Z)-heptenoicacid;(.+−.)(5Z)-7-[3-endo-((phenylsulfonyl)amino]-bicyclo[2.2.1]hept-2-exo-yl]-heptenoicacid (S-1452, Shionogi domitroban. Anboxan®.);(−)6,8-difluoro-9-p-methylsulfonylben-zyl-1,2,3,4-tetrahydrocarbazol-1-yl-aceticacid (L670596, Merck) and(3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]-2,2-dimethylpropanoicacid (L655240, Merck).

The preferred thromboxane A2 receptor antagonist of the presentinvention is ifetroban or any pharmaceutically acceptable salts thereof.

In certain preferred embodiments the preferred thromboxane A2 receptorantagonist is ifetroban sodium (known chemically as[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt.

Methods of Treatment

In certain embodiments of the present invention there is provided amethod of treating and/or ameliorating cardiomyopathies in a patient orpatient population by administration of a therapeutically effectiveamount of a thromboxane A₂ receptor antagonist to a patient(s) in needthereof.

The administration of a therapeutically effective amount of athromboxane A₂ receptor antagonist may be accomplished via anytherapeutically useful route of administration, including but notlimited to orally, intranasally, rectally, vaginally, sublingually,buccally, parenterally, or transdermally. In certain preferredembodiments, the thromboxane A₂ receptor antagonist is administeredparenterally. In certain further embodiments, the thromboxane A₂receptor antagonist is administered by intra-articular injection. Incertain further embodiments, the thromboxane A₂ receptor antagonist isadministered directly to the affected anatomic site. In anotherembodiment, the thromboxane A₂ receptor antagonist is administeredthrough the hepatic artery.

In certain preferred embodiments, the plasma concentrations ofthromboxane A₂ receptor antagonists range from about 0.1 ng/ml to about10,000 ng/ml. Preferably, the plasma concentration of thromboxane A₂receptor antagonists range from about 1 ng/ml to about 1,000 ng/ml.

When the thromboxane A₂ receptor antagonists is ifetroban, the desiredplasma concentration for treatment of cardiomyopathies in musculardystrophies in certain embodiments should be greater than about 10 ng/mL(ifetroban free acid). Some therapeutic effects of thromboxane receptorantagonist, e.g., ifetroban, may be seen at concentrations of greaterthan about 1 ng/mL.

The dose administered should be adjusted according to age, weight andcondition of the patient, as well as the route of administration, dosageform and regimen and the desired result.

In order to obtain the desired plasma concentration of thromboxanereceptor antagonists for the treatment of cardiomyopathy in musculardystrophy patients, daily doses of the thromboxane A₂ receptorantagonists preferably range from about 0.1 mg to about 5000 mg. Incertain preferred embodiments, the thromboxane A₂ receptor antagonist isadministered on a chronic basis. Daily doses may range from about 1 mgto about 1000 mg; about 10 mg to about 1000 mg; about 50 mg to about 500mg; about 100 mg to about 500 mg; about 200 mg to about 500 mg; about300 mg to about 500 mg; or from about 400 mg to about 500 mg per day. Incertain preferred embodiments where the mammal is a human patient, thetherapeutically effective amount is from about 100 mg to about 2000 mgper day, or from about 10 mg or about 100 mg to about 1000 mg per day,and certain embodiments more preferably from about 50 to about 500 mgper day, or from about 100 mg to about 500 mg per day. The daily dosemay be administered in divided doses or in one bolus or unit dose or inmultiple dosages administered concurrently. In this regard, theifetroban may be administered orally, intranasally, rectally, vaginally,sublingually, buccally, parenterally, or transdermally. In certainpreferred embodiments, the pharmaceutical composition described above,the therapeutically effective amount is from about 10 mg to about 1000mg ifetroban (or pharmaceutically acceptable salt thereof) per day. Incertain preferred embodiments, the therapeutically effective amount isfrom about 100 to about 500 mg per day, and in certain embodiments fromabout 150 mg to about 350 mg per day will produce therapeuticallyeffective plasma levels of ifetroban free acid for the treatment ofmuscular dystrophy. In certain preferred embodiments, a daily dose ofifetroban sodium from about 10 mg to about 250 mg (ifetroban free acidamounts) will produce therapeutically effective plasma levels ofifetroban free acid for the treatment of muscular dystrophy.

Preferably, the therapeutically effective plasma concentration ofthromboxane A₂ receptor antagonists ranges from about 1 ng/ml to about1,000 ng/ml for the treatment of muscular dystrophy.

When the thromboxane A₂ receptor antagonist is ifetroban, the desiredplasma concentration for providing an inhibitory effect ofA₂/prostaglandin endoperoxide receptor (TPr) activation, and thus areduction of cerebral microvascular activation should be greater thanabout 10 ng/mL (ifetroban free acid). Some inhibitory effects ofthromboxane A₂ receptor antagonist, e.g., ifetroban, may be seen atconcentrations of greater than about 1 ng/mL.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result.

However, in order to obtain the desired plasma concentration ofthromboxane A₂ receptor antagonists, daily doses of the thromboxane A₂receptor antagonists ranging from about 0.1 mg to about 5000 mg shouldbe administered. Preferably, the daily dose of thromboxane A₂ receptorantagonists ranges from about 1 mg to about 1000 mg; about 10 mg toabout 1000 mg: about 50 mg to about 500 mg; about 100 mg to about 500mg; about 200 mg to about 500 mg; about 300 mg to about 500 mg; andabout 400 mg to about 500 mg per day.

In certain preferred embodiments, a daily dose of ifetroban sodium fromabout 10 mg to about 250 mg (ifetroban free acid amounts) will produceeffective plasma levels of ifetroban free acid.

Pharmaceutical Compositions

The thromboxane A₂ receptor antagonists of the present invention may beadministered by any pharmaceutically effective route. For example, thethromboxane A₂ receptor antagonists may be formulated in a manner suchthat they can be administered orally, intranasally, rectally, vaginally,sublingually, buccally, parenterally, or transdermally, and, thus, beformulated accordingly.

In certain embodiments, the thromboxane A₂ receptor antagonists may beformulated in a pharmaceutically acceptable oral dosage form. Oraldosage forms may include, but are not limited to, oral solid dosageforms and oral liquid dosage forms.

Oral solid dosage forms may include, but are not limited to, tablets,capsules, caplets, powders, pellets, muitiparticulates, beads, spheresand any combinations thereof. These oral solid dosage forms may beformulated as immediate release, controlled release, sustained(extended) release or modified release formulations.

The oral solid dosage forms of the present invention may also containpharmaceutically acceptable excipients such as fillers, diluents,lubricants, surfactants, glidants, binders, dispersing agents,suspending agents, disintegrants, viscosity-increasing agents,film-forming agents, granulation aid, flavoring agents, sweetener,coating agents, solubilizing agents, and combinations thereof.

Depending on the desired release profile, the oral solid dosage forms ofthe present invention may contain a suitable amount ofcontrolled-release agents, extended-release agents, modified-releaseagents.

Oral liquid dosage forms include, but are not limited to, solutions,emulsions, suspensions, and syrups. These oral liquid dosage forms maybe formulated with any pharmaceutically acceptable excipient known tothose of skill in the art for the preparation of liquid dosage forms.For example, water, glycerin, simple syrup, alcohol and combinationsthereof.

In certain embodiments of the present invention, the thromboxane A₂receptor antagonists may be formulated into a dosage form suitable forparenteral use. For example, the dosage form may be a lyophilizedpowder, a solution, suspension e.g., depot suspension).

In other embodiments, the thromboxane A₂ receptor antagonists may beformulated into a topical dosage form such as, but not limited to, apatch, a gel, a paste, a cream, an emulsion, liniment, balm, lotion, andointment.

Detailed Description of the Preferred Embodiments

The following examples are not meant to be limiting and representcertain embodiments of the present invention.

Example I

In this example, ifetroban sodium tablets are prepared with thefollowing ingredients listed in Table 1:

TABLE 1 Ingredients Percent by weight Na salt of Ifetroban 35 Mannitol50 Microcrystalline Cellulose 8 Crospovidone 3.0 Magnesium Oxide 2.0Magnesium Stearate 1.5 Colloidal Silica 0.3

The sodium salt of ifetroban, magnesium oxide, mannitol,microcrystalline cellulose, and crospovidone is mixed together for about2 to about 10 minutes employing a suitable mixer. The resulting mixtureis passed through a #12 to #40 mesh size screen. Thereafter, magnesiumstearate and colloidal silica are added and mixing is continued forabout 1 to about 3 minutes.

The resulting homogeneous mixture is then compressed into tablets eachcontaining 35 mg, ifetroban sodium salt.

Example II

In this example, 1000 tablets each containing 400 mg of Ifetroban sodiumare produced from the following ingredients listed in. Table 2:

TABLE 2 Ingredients Amount Na salt of Ifetroban 400 gm Corn Starch 50 gGelatin 7.5 g Microcrystalline Cellulose (Avicel) 25 g MagnesiumStearate 2.5 g

Example III

An injectable solution of ifetroban sodium is prepared for intravenoususe with the following ingredients listed in Table 3:

TABLE 3 Ingredients Amount Ifetroban Sodium 2500 mg Methyl Paraben 5 mgPropyl Paraben 1 mg Sodium Chloride 25,000 mg Water for injection q.s. 5liter

The sodium salt of ifetroban, preservatives and sodium chloride aredissolved in 3 liters of water for injection and then the volume isbrought up to 5 liters. The solution is filtered through a sterilefilter and aseptically filled into pre-sterilized vials which are thenclosed with pre-sterilized rubber closures. Each vial contains aconcentration of 75 mg of active ingredient per 150 ml of solution.

Example IV

dSG KO mice, chosen for their cardiac phenotype, are a model of LGMD,but DMD which occurs in approximately 1:3500 male births (1), is farmore common a disease than LGMD. The mdx mouse model of DMD poorlyreplicates the shortened life expectancy, cardiac fibrosis, andcardiomyopathy seen in DMD patients. The utrophin/dystrophin DKO modelhad significant mortality by 10 weeks, although treatment with the TPrantagonist ifetroban led to 100% survival to this predeterminedtimepoint. Although TPr antagonism may prevent spontaneous death in DMD,due to severe kyphosis and frailty we were not able to obtain muchuseful cardiac data with the DKO model of DMD.

Example 4 utilized West/Carrier Muscular Dystrophy Animal Models(Delta-sarcoglycan knock-out mice (sgcd−/−)). Mice devoid of DSG developcardiomyopathy and MD with signs of progressive disease such asnecrosis, muscular regeneration, inflammation and fibrosis within thefirst 3 months of life. Mice that are homozygous for the targetedmutation are viable, fertile and normal in size. No gene product(protein) is immunodetected in skeletal muscle microsomal preparations.At 8 weeks of age there is an onset of sudden mortality, with a 50%survival rate at 28 weeks. Elevated creatine kinase serum levels areindicative of striated muscle degeneration. Histopathology of skeletalmuscle tissue reveals degeneration and regeneration of muscle fibers,inflammatory infiltrate, perivascular fibrosis and calcification. At 12weeks of age, cardiac muscle tissue also begins to show degeneration,inflammatory infiltration and perivascular fibrosis. Myofiber membraneshave permeability defects as assessed by Evans blue dye uptake intomyofiber cytoplasm. Skeletal muscle of mutant mice have an enhancedsensitivity to mechanically induced sarcolemmal damage. Dystrophindeficient mice have minimal clinical symptoms with lifespan reduced byonly 25% unlike humans with DMD reduced by 75%, possibly due tocompensatory mechanisms upregulated in mice. A major function ofdystrophin is to strengthen the sarcolemma by cross-linking the ECM withthe cytoskeleton. Utrophin and a7b1 integrin fulfil the same functionand are upregulated in mdx mice. They work to connect sarcolemma tocytoplasmic actin cytoskeleton. Dysfunction produces membraneinstability, elevated [Ca2+]I and disrupted NO signaling γ- and δ-SGform a core necessary for delivery/retention of other SG to themembrane.

While the DSG KO (sgcd−/−) mice lack functional delta-sarcoglycan, theMD phenotype is milder than the human disease. Since utrophin, adystrophin-related protein, is able to compensate for the loss ofdystrophin, loss of utrophin and dystrophin (DKO) results in a moresevere phenotype. DKO are significantly smaller and show more severemuscle disease (similar or worse than that of humans with MD). The miceare difficult to generate and care for, and often die prematurely.Ifetroban treatment was started at 3 weeks upon weaning.

In Example IV, vehicle-treated mice were carefully cared for to get themto reach 10 weeks of life (e.g., the mice were checked on themconstantly and a low dish of crushed food and water was placed rightnext to where the mice huddled in the cage, in an attempt to get themsome nutrition without them needing to move much).

FIG. 1 are photos of a vehicle-treated compared with anifetroban-treated DKO mouse. FIG. 1A is a photograph of avehicle-treated DKO Mouse at 10 weeks. FIG. 1B is a photograph of anifetroban-treated DKO mouse at 10 weeks. The ability to wrap the tailaround the wire is dependent on muscle function. A reason the DKO miceare really hard to evaluate in the wire hang is that they have suchsevere scoliosis that their hind paws are very close to their frontpaws, so raising their hind paws to get a 4-limbed grip is not difficultdespite their affliction.

FIG. 2 shows plasma cTNI in dSG KO males at 3 months. The term “cTNI”means plasma cardiac troponin I. The term “KO” means knockout. The term“dSG” means Delta sarcoglycan. The term “WT” means wildtype. Plasmacardiac troponin I (cTNI) is highly specific and sensitive formyocardial tissue and can be measured rapidly. It is a reliablebiomarker for cardiac damage. In FIG. 2, it can he seen that the plamscTNI levels are much higher in dSG KO mice than in WT mice.

FIG. 3 provides 3 month Echo data. The results shown therein demonstratethat at 3 months dSG males show cardiac dysfunction and ifetrobanprevents cardiac dysfunction.

FIG. 4 provides cardiac output data for male dSG KO mice at 3 months.FIG. 4 shows that the dSG KO mice treated with ifetroban have improvedcardiac dysfunction compared to vehicle. The cardiac function improvedsimilar to WT levels.

FIG. 5 provides spontaneous exercise date for 6 month old males. Theexercise was voluntary wheel running-free access to the wheel for 10days after 4.5M of treatment. Males demonstrate a skeletal functiondeficit at 6M that is seen to a less extent in ifetroban-treated DSG KOmice. No difference is seen in females who run more compared to malesregardless of genotype.

FIG. 6 shows wire hang in dSG mice at 6 months. An improved wire hangtime is apparent in the dSG mice treated with ifetroban. *p<0.05 from WTby one-way ANOVA followed by Dunnett's multiple comparison post-test.Veh and ife-treated groups were NS tested against each other. N inparentheses. “ife”=ifetroban.

FIG. 7 shows the results of a wire hanging experiment at 6 months, withthe average hang time plotted for dSG and WI mice.

FIG. 8 depicts wire hang time for mice tested. Male mice do not hang fora long time compared to females. It was difficult to measure anydifference caused by ifetroban if any.

FIGS. 9A (dSGKO-vehicle) and 9B (DsGKO-ifetroban) show cardiac histologyin dSG KO males. Less fibrosis seen in ifetroban treated RV. Shown isMasson's trichrome at 4× for gross histology. All tears/folds/redhotspots from slice preparation and not pathology. Some RV may also beaffected by slicing (arrows).

FIGS. 10A(dSG-Veh), 10B(dSG-veh), 10C(dSG-ifetroban) and10D(dSG-ifetroban) show cardiac histology in dSG KO males (usingMasson's trichrome, 2×). It can be seen that there is less fibrosis inthe ifetroban treated RV. RV=right ventricle.

FIGS. 11A1, 11A2, 11A3 and 11A4 shows cardiac histology in dSG KO males(using Masson's trichrome, 10×) in the left ventricle (11A1=mouse #1,dSG KO-vehicle; 11A2=mouse #2, dSG KO-vehicle; 11A3=mouse #1, dSGKO-ifetroban; and 11A4=mouse #2, dSG KO-ifetroban); FIGS. 11B1, 11B2,11B3 and 11B4 shows cardiac histology in the right ventricle (11B1=mouse#1, dSG KO vehicle: 11B2=mouse #2, dSG KO-vehicle; 11B3=mouse #1. dSGKO-ifetroban; and 11B4=mouse #2, dSG KO-ifetroban). LV=left ventricle;RV=right ventricle. Less fibrosis was seen in ifetroban-treated KO mice.

FIGS. 12A(WT1), 12B(dSG-KG-vehicle), 12C(WT2) and 12D(dSG-KO-ifetroban)shows skeletal muscle histology in WT and dSG KO males (tibialiscross-section, using Masson's trichrome). Some fibrosis may be due tospecific section of muscle.

FIGS. 13A(WT-vehicle), 13B(WT-ifetroban), 13C(dSG KO-vehicle) and13D(dSG-KO-ifetroban) are cross-sections of intestinal tissue showingthat ifetroban may prevent the loss of intestinal smooth muscle in thelarge intestine Muscularis. The DSG KO mice were missing smooth muscle(especially missing longitudinal smooth muscle) while ifetroban-treatedmice have similar sections to WT smooth muscle. “H&E” Hematoxylin &eosin. FIG. 13 shows that ifetroban-treated dSG KO mice have lessfibrosis than vehicle-treated dSG KO mice.

FIGS. 14A and 14B are graphs showing the percent survival of dSG KOmales (14A) and dSG females (14B) treated with ifetroban or vehicle.

FIG. 15 are graphs showing wire hang in DKO males at 10 weeks(ifetroban-treated (“ife”) versus vehicle). The results show that theifetroban-treated mice had significantly longer average hang times thanmice treated with vehicle.

FIG. 16 shows spontaneous running in DKO mice: measured from 9-10 weeks.

FIG. 17 shows survival for all DKO mice. The ifetroban-treated micesurvived beyond 70 days, while the vehicle-treated mice (both male andfemale) did not.

CONCLUSION

In the preceding specification, the invention has been described withreference to specific exemplary embodiments and examples thereof. Itwill, however, be evident that various modifications and changes may bemade thereto without departing from the broader spirit and scope of theinvention as set forth in the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative manner ratherthan a restrictive sense.

What is claimed is:
 1. A method of treating or ameliorating musculardystrophy in a subject in need of treatment thereof, comprisingadministering a therapeutically effective amount of a thromboxane A₂receptor antagonist to the patient.
 2. The method of claim 1, whereinthe muscular dystrophy is fibrosis is selected from the group consistingof Duchenne MD (DMD), Becker MD, and Limb-Girdle MD.
 3. The method ofclaim 1, further comprising administering the thromboxane A₂ antagonistto the patient on a chronic basis.
 4. The method of claim 3, wherein thecardiac function of the patient is maintained or improved.
 5. The methodof claim 3, wherein the thromboxane A₂ receptor antagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid (Ifetroban), and pharmaceutically acceptable salts thereof.
 6. Themethod of claim 3, wherein the thromboxane A₂ receptor antagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoic acid, monosodiumsalt (Ifetroban Sodium).
 7. The method of claim 1, wherein thethromboxane A₂ receptor antagonist is administered orally, intranasally,rectally, vaginally, sublingually, buccally, parenterally, ortransdermally.
 8. The method of claim 1, wherein the thromboxane A₂receptor antagonist is administered parenterally.
 9. The method of claim1, wherein the thromboxane A₂ receptor antagonist is administeredorally.
 10. The method of claim 3, wherein the thromboxane A₂ receptorantagonist is administered prophylactically to prevent cardiomyopathy inthe patient.
 11. The method of claim 3, wherein the thromboxane A₂receptor antagonist is administered prophylactically to preventgastrointestinal dysfunction in the patient.
 12. The method of claim 3,wherein the therapeutically effective amount is from about 50 mg toabout 500 mg.
 13. The method of claim 5, wherein the therapeuticallyeffective amount is from about 150 mg to about 350 mg per day and theifetroban is administered orally.
 14. A method of treating cardiacand/or gastrointestinal dysfunction in a human patient suffering frommuscular dystrophy, comprising chronically administering atherapeutically effective amount of a thromboxane A₂ receptor antagonistto the human patient.
 15. The method of claim 14, wherein thetherapeutically effective amount is from about 100 mg to about 500 mg.16. The method of claim 14, wherein the thromboxane A₂ receptorantagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]benzenepropanoicacid (Ifetroban), and pharmaceutically acceptable salts thereof.
 17. Themethod of claim 16, wherein the thromboxane A₂ receptor antagonist is[1S-(1α,2α,3α,4α)]-2-[[3-[4-[(Pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl]-benzenepropanoicacid, monosodium salt (Ifetroban Sodium).
 18. The method of claim 16,wherein the therapeutically effective amount is from about, 150 mg toabout 350 mg per day and the ifetroban is administered orally.
 19. Themethod of claim 13, wherein the gastrointestinal dysfunction is smoothmuscle dysfunction.
 20. The method of claim 16, wherein thetherapeutically effective amount of ifetroban provides improvedventricular function to the heart of the patient.